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WO1994022848A1 - Analogues d'hepoxiline utilisables comme agents anti-inflammatoires - Google Patents

Analogues d'hepoxiline utilisables comme agents anti-inflammatoires Download PDF

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Publication number
WO1994022848A1
WO1994022848A1 PCT/IB1994/000085 IB9400085W WO9422848A1 WO 1994022848 A1 WO1994022848 A1 WO 1994022848A1 IB 9400085 W IB9400085 W IB 9400085W WO 9422848 A1 WO9422848 A1 WO 9422848A1
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Prior art keywords
alkyl
compound according
double
hepoxilin
mixture
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PCT/IB1994/000085
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English (en)
Inventor
Cecil R. Pace-Asciak
Peter M. Demin
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Pace Asciak Cecil R
Demin Peter M
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Application filed by Pace Asciak Cecil R, Demin Peter M filed Critical Pace Asciak Cecil R
Priority to AU65117/94A priority Critical patent/AU6511794A/en
Priority to DE69411716T priority patent/DE69411716T2/de
Priority to EP94912660A priority patent/EP0691963B1/fr
Publication of WO1994022848A1 publication Critical patent/WO1994022848A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D331/00—Heterocyclic compounds containing rings of less than five members, having one sulfur atom as the only ring hetero atom
    • C07D331/02—Three-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/27—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation
    • C07C45/29—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation of hydroxy groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C45/00—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds
    • C07C45/27—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation
    • C07C45/29—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation of hydroxy groups
    • C07C45/292—Preparation of compounds having >C = O groups bound only to carbon or hydrogen atoms; Preparation of chelates of such compounds by oxidation of hydroxy groups with chromium derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C47/00—Compounds having —CHO groups
    • C07C47/38—Unsaturated compounds having —CHO groups bound to carbon atoms of rings other than six—membered aromatic rings
    • C07C47/45—Unsaturated compounds having —CHO groups bound to carbon atoms of rings other than six—membered aromatic rings having unsaturation outside the rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/66—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety
    • C07C69/73—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids
    • C07C69/732—Esters of carboxylic acids having esterified carboxylic groups bound to acyclic carbon atoms and having any of the groups OH, O—metal, —CHO, keto, ether, acyloxy, groups, groups, or in the acid moiety of unsaturated acids of unsaturated hydroxy carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D303/00—Compounds containing three-membered rings having one oxygen atom as the only ring hetero atom
    • C07D303/02—Compounds containing oxirane rings
    • C07D303/38—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D303/40—Compounds containing oxirane rings with hydrocarbon radicals, substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals by ester radicals
    • C07D303/42—Acyclic compounds having a chain of seven or more carbon atoms, e.g. epoxidised fats

Definitions

  • Second messengers play an important role in maintaining homeostasis in a diverse number of metabolic processes.
  • Calcium is an important member of the group of second messengers, and regulation of calcium has become a focal point for investigating and controlling metabolic pathways and pathological conditions that can result from the aberrant regulation of these pathways. Regulation is achieved by opening or closing gated ion channels. This results in a change in the intracellular ion concentration in either of two ways: 1) changing the voltage across the plasma membrane or 2) allowing a major influx of ions, both generating an intracellular response.
  • Calcium-regulated cell signaling pathways regulate cellular functions such as inflammation and smooth muscle contraction.
  • Inflammation is the body's reaction to injury.
  • the inflammatory response involves three stages: first, an increase of blood flow to the injured area; second, an increase of capillary permeability caused by the retraction of endothelial cells lining vessel walls; and third, leucocyte migration to the site of injury.
  • the third stage known as chemotaxis, is a complex process that results in phagocytosis of invading agents by certain types of leucocytes such a ⁇ the neutrophil.
  • the neutrophil plays a key role in the body's response in inflammatory events such as infection.
  • the neutrophil Once having arrived at the site of inflammation, the neutrophil is "activated” and releases a plethora of oxidative enzymes, known as a respiratory burst, that aid in destroying the invasive agent.
  • a respiratory burst oxidative enzymes
  • An increase in intracellular calcium is thought to be involved in the initiation of the events that result in respiratory burst.
  • Hepoxilins are products of an arachidonic acid pathway and have been implicated in the mediation of inflammation and smooth muscle contraction by modulation of second messenger calcium in response pathways. Hepoxilins are biologically active hydroxy epoxide derivatives of arachidonic acid formed through the 12-lipoxygenase pathway (Pace-Asciak et al., J. Biol. Chem. 258: 6835-6840, 1983; Pace-Asciak, Biochim. Biophys.
  • Hepoxilin A3 [8(S, R)-hydroxy-11(S) ,12 (S)- epoxy-eicosa-5Z, 9E, 14Z-trienoic acid] and hepoxilin B3 [10(S, R)-hydroxy-ll(S) ,12 (S)-epoxy-eicosa-5Z, 8Z, 14Z- trienoic acid] .
  • the term "hepoxilin” was coined in an attempt to combine aspects of structure with their first, though not necessarily their most important, demonstrated biological activity of insulin release (Pace-Asciak and Martin, Prost l. Leukotriene and Med. 16: 173-180, 1984).
  • Hepoxilin A3 epimers are represented as:
  • Hepoxilin B3 epimers are represented as:
  • Hepoxilins are probably formed wherever 12- lipoxygenase is present because 12-HPETE is actively transformed into the hepoxilins by a variety of ferriheme proteins. Hence, ferriprotoporphyrin and such containing groups in proteins catalyze this transformation (Pace- Asciak et al., Bioloc-f. Oxidation Systems, Eds C.C. Reddy et al., Academic Press, NY, 725-735, 1990) .
  • Hepoxilins are formed by platelets (Bryant and Bailey, Prostacrlandins 17: 9-18, 1979; Jones et al., Prostacrlandins 16: 583-590, 1978), lung (Pace-Asciak et al., Biochi . Biophvs. Acta 712: 142-145, 1982), pancreatic islets (Pace-Asciak and Martin, ibid. 1984) , brain (Pace-Asciak, ibid. 1988) , aorta (Laneuville et al., Biochim. Biophvs. Acta 1084: 60-68, 1991) and neutrophils (Dho et al., Biochem. J.
  • Hepoxilin B3 has been isolated from marine red algae (Moghaddam et al., J. Biol. Chem. 265: 6126-6130, 1990) and hepoxilin A3 has been detected in the Aplysia brain (Piomelli et al., Proc. Natl. Acad. Sci. USA 86: 1721- 1725, 1989) . Hepoxilins are also formed by the rat pineal gland (Reynaud et al., J. Neurochem. 62: 126-133, 1994) . Hepoxilins have been shown to possess a variety of biological actions related to their ability to affect ion fluxes in the cell.
  • Hepoxilins raise intracellular calcium in human neutrophils (Dho et al., Biochem. J. 266:63-68, 1990), increase the transport of calcium across membranes (Derewlany et al., Can. J. Physiol. Pharmacol. 62: 1466-1469, 1984), stimulate the release of insulin (Pace-Asciak and Martin, ibid. 1984) , and regulate the volume of human platelets through an effect on potassium channels in the cell (Margalit et al. 1993 Proc. Natl. Acad. Sci. USA £fl: 2589-2592, 1993).
  • Biological actions of the hepoxilins demonstrated so far include the potentiation of aortic and tracheal vasoconstriction (Laneuville et al, Br. J. Pharmacol. 105: 297-304, 1992; 107: 808-812, 1992), potentiation of vascular permeability (Laneuville and Pace-Asciak, Prostaqlandins, Leukotrienes. Lipoxins and PAF. Ed. J.M. Bailey, Plenum Press NY: 335-338, 1991), modulation of second messenger systems (Niga et al., Biochem. Biophys. Res. Co . 171: 944-948, 1990), regulation of cell volume (Margalit et al. Proc.
  • Hepoxilin antagonists find utility in reducing inflammation, asthma, hypertension, migraine and septic shock and in modulating other processes mediated by cellular calcium levels. Hepoxilin agonists find utility in diabetes.
  • Figure 1 illustrates a synthetic scheme for cyclopropyl hepoxilin analogs.
  • Figure 2 illustrates a synthetic scheme for thiirano hepoxilin derivatives.
  • Figure 3 illustrates a synthetic scheme for radiolabeling hepoxilin.
  • Figure 4 illustrates hepoxilin analogs compete for binding on neutrophils with tritiated hepoxilin.
  • the present invention provides hepoxilin analogs that modulate the mobilization of intracellular calcium in human neutrophils induced by such agonists as f-Met-Leu-Phe (fMLP) , platelet activation factor (PAF) , leukotriene B 4 (LTB4) , hepoxilin A 3 and thapsigargin.
  • fMLP f-Met-Leu-Phe
  • PAF platelet activation factor
  • LTB4 leukotriene B 4
  • hepoxilin A 3 thapsigargin.
  • Certain of these analogs have been found to antagonize hepoxilin activity in experimental models. As such they may be useful in the modulation of hepoxilin-mediated (or hepoxilin agonist-mediated) processes, including inflammation associated with neutrophil activation in inflammatory disease.
  • the present invention is directed to hepoxilin analogs having the general structure I or II:
  • X is O, C n , NH, or S, wherein n is 1, 2, 3 or 4; Rl is OH, CH 3 , CH 2 OH, N 3 or CH 2 N 3 ; R3 is H or CH 3 ; R5 is Y-R2, wherein Y is a six- carbon chain optionally containing up to three double or triple bonds or a mixture of double and triple bonds up to a maximum of three; R2 i ⁇ C ⁇ -C ⁇ alkyl—OH, C .-C 10 alkyl—N 3 or C00R4, wherein R4 is H, a branched or unbranched C ] _-C ⁇ 0 alkyl (including substituted alkyl radicals) , cycloalkyl (including substituted cycloalkyl) , preferably C5 or C5 cycloalkyl, or a five- or six- membered aryl radical (including substituted aryl radicals), i.e.
  • R2 is COOH or an ester of R4;
  • R6 is a seven-carbon chain optionally containing up to three double or triple bonds or a mixture of double and triple bonds up to a maximum of three; and indicate ⁇ a single, double or triple bond; subject to the limitation that when the structure is
  • hepoxilin analogs of the present invention are of the following structures III-IV:
  • Rl, R2, R3 , R4 and X are as previously defined, subject to the limitation that if Rl is OH, R2 is COOH or COOCH 3 , and R3 is H, then X is not O.
  • Preferred substitutions within R4 include lower alkyl and halo.
  • alkyl is used herein to refer to branched and unbranched alkyl radical ⁇ .
  • Suitable alkyl radical ⁇ include methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, n-amyl, sec- amyl, n-hexyl, 2-ethylbutyl, 2,3-dimethylbutyl and the like.
  • Lower alkyl C__-- * Q alkyl) radicals are preferred, such as methyl and ethyl radicals.
  • alkenyl refers to branched and unbranched hydrocarbon chains having one or more double bonds, including mono- un ⁇ aturated and poly-un ⁇ aturated radicals. Lower alkenyl radicals having from one to six carbon atom ⁇ are preferred.
  • alkynyl refers to branched and unbranched hydrocarbon chains having one or more triple bonds, including mono-unsaturated and poly-unsaturated radicals. Lower alkynyl radicals having from one to six carbon atoms are preferred.
  • aryl include ⁇ monocyclic aromatic hydrocarbon radical ⁇ . Suitable aryl groups include phenyl, chlorophenyl, benzyl and the like.
  • halo includes chloro, fluoro, bromo and iodo. The ⁇ ymbol i ⁇ u ⁇ ed to indicate a bond that can be single, double or triple.
  • Preferred epimers of hepoxilin analogs III-VI include the following:
  • X is preferably NH, S or C n , wherein n is 1, 2, 3 or 4; Rl is OH, CH 3 , CH 2 0H, N 3 or CH 2 N 3 ; R2 is COOH or COOCH 3 . and R3 is H, CH 3 OR CH 2 OH.
  • Rl is OH
  • X and carbon atoms 11 and 12 will form a three- to six-membered ring, optionally containing one or more double bonds, or an aromatic ring.
  • Rl is OH
  • X is C n , wherein n is most preferably 1, forming a cyclopropane ring
  • R3 is H.
  • X is C n , NH or S; Rl is OH; R2 is COOR4; R3 is H or CH 3 ; and R4 is H or lower alkyl, particularly methyl.
  • X is O and R2 is alkyl—N 3 , alkyl—OH, an ester of a C 5 ⁇ C 10 alkyl radical, or an ester of an aryl radical, particularly an ester of a substituted or unsubstituted phenyl radical.
  • Particularly preferred molecules within this group are tho ⁇ e in which R2 is alkyl—N 3 or alkyl— OH, such as lower alkyl—N 3 or lower alkyl—OH.
  • structural analogs of hepoxilin ⁇ including the representative hepoxilin antagonists methyl 8-hydroxy-ll,12-cyclopropyl- 5(Z) ,9(E) ,14(Z)-eico ⁇ atrienoate ( ⁇ HXA 3 ) and methyl 10- hydroxy-ll,12-cyclopropyl-5(Z) ,8(Z) ,14(Z)-eicosatrienoate ( ⁇ HXB 3 ) , may conveniently be synthesized utilizing the acetylenic approach to hepoxilins (Demin et al., Bioorg. Khim 16:1125-1133. 1990).
  • a ⁇ ynthetic ⁇ cheme for hepoxilin antagoni ⁇ ts is set forth in Figure 1.
  • 1- hydroxyundeca-2(E)-en-5-yne (1) a common intermediate in this synthetic scheme, prepared as described (Demin et al., ibid.), is modified to obtain the corresponding three-membered cyclopropane ring- containing alcohols, which are the key synthon ⁇ to hepoxilin analogs.
  • alcohol (1) [1-Hydroxyundeca- 2(E)-en-5-yne] is treated with CH 2 I and Zn-Cu couple in dry ether giving racemic (2S*,3S*)-2,3-cyclopropylalcohol (2) , which is then oxidized to aldehyde (3) with pyrydiniu dichromate.
  • Two subsequent condensation ⁇ of aldehyde (3) with Li-derivative of propargyl chloride leads to cyclopropylcarbinol (4) .
  • 1 H-NMR spectrum at this stage shows a 7:3 ratio between two diastereomers.
  • NMR spectra show the two differences between epimers: coupling constant J ⁇ o, ll ⁇ larger for the more polar epimer (d, 7.8 Hz) than for the less polar epimer (d, J 7.3 Hz), and proton at C 11 of cyclopropyl group has less chemical shift (0.68 ppm) for the more polar epimer than for the less polar epimer (0.81 ppm) .
  • the more polar ⁇ HXA3 (obtained from anti ⁇ HXB3) is referred to as syn or (8R*, IIS*, 12S*) -epimer (7b), and the les ⁇ polar ⁇ HXA3 a ⁇ anti or (8S*, IIS*, 12S*)-epimer (7a) , re ⁇ pectively.
  • the chromatographic properties of ⁇ HXA3 methyl esters (7a) , (7b) are also similar to native HXA3 methyl esters with known relative configuration (Demin et al., Bioorg. Khim..
  • hepoxilin analogs may be prepared in the form of pharmaceutically acceptable salts, especially acid-addition salt ⁇ , including salts of organic acids and mineral acids.
  • salts include organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, tris hydroxy amino methyl (THAM) and the like.
  • Suitable inorganic acid-addition ⁇ alt ⁇ include salts of hydrochloric, hydrobromic, sulfuric and pho ⁇ phoric acids and the like.
  • the acid addition salts may be obtained as the direct products of compound synthe ⁇ is.
  • the free base may be di ⁇ solved in a suitable solvent containing the appropriate acid, and the salt isolated by evaporating the solvent or otherwise separating the salt and solvent.
  • Hepoxilin antagonists may be administered for the treatment of such conditions as inflammation, asthma, hypertension, migraine and septic shock.
  • the compounds or their salts are formulated for enteral or parenteral administration by combining them with a pharmaceutically acceptable vehicle according to conventional procedures. See, Remington's Pharmaceutical Science ⁇ . 18th ed. , Gennaro, ed.. Mack Publishing Co, Easton, PA, 1990, incorporated herein by reference.
  • the active ingredient will be combined with one or more diluents, fillers, emul ⁇ ifier ⁇ , excipient ⁇ , etc., and may be provided in ⁇ uch form ⁇ a ⁇ liquid ⁇ , powder ⁇ , emul ⁇ ions, tablets, cap ⁇ ule ⁇ and the like.
  • Such compo ⁇ itions may further include one or more auxiliary substances, including wetting agents, preservatives, stabilizers, buffers, lubricants, colorings, etc.
  • GC analyses were carried out on a Hewlett-Packard 5700A gas chromatograph (Hewlett-Packard, Palo Alto, CA) using a glas ⁇ capillary column, (SPB-1, Supelco, Beliefonte, PA) 60 m x 0.3 mm. Electron impact ma ⁇ ⁇ pectra were obtained on a Hewlett- Packard GC MS u ⁇ ing a fu ⁇ ed ⁇ ilica methyl silicone capillary column (HP-1, 12 m x 0.2 mm).
  • Mass- spectrum (m/z, % of related intensity): 177 ([M - 1] + , 0.4), 149 ([M - CH0] + , 5.0), 121 ([M - C 4 H 9 ] " , 10), 107 ([M - C 5 Hn] + , 25), 93 ([M - C 4 H 9 - CHO + H]+, 29), 79 ([M - C 5 H1 1 - CHO + H] + , 47), 69 (100).
  • Mass spectrum of the tBDMSi derivative (m/z, % of related intensity): 365 ([M - 1] + , 0.15), 331 ([M - Cl] + , 1.9), 309 ( [M - t-Bu] + , 5.0), 275 ([M - t-Bu - Cl + H] + , 6.8), 199 ( [M - Cl - tBDMSi] + , 24), 129 ([M - C5H11 - Cl - t-BuMe2SiOH + H] + , 92), 128 (100) .
  • Mass spectrum of the tBDMS derivative (m/z, % of related intensity) : 455 ( [M - 1] + , 0.15), 425 ([M - OMe]+, 0.19), 399 ( [M - t-Bu] + , 37), 293 ([C 10 -C 20 ]+, 3.7), 317 ([C 8 -C 20 ] + , 0.90), 307 ([C ⁇ -C 10 ] , 0.80), 221 (64), 189 ([(C ⁇ -C 11 ) - t-BuMe2SiOH + H] + , 17), 75 (100) .
  • reaction mixture wa ⁇ analyzed by GC-MS as the tBDMSi derivative monitoring the prominent ion m/z 405 [M - t-Bu] + .
  • GC MS analysis of the reaction mixture showed 92% conversion into the desirable trienes (6a, b) .
  • the catalyst was filtered off, the filtrate was poured onto an aluminum oxide column (pH 6.9-7.1) . Quinoline was removed with 5% EtOAc in hexane.
  • Mass spectrum, tBDMSi derivative (m/z, % of related intensity) : 462 ([M] + , 0.06), 431 ([M - OMe] + , 0.80), 405 ( [M - t-Bu] + , 37), 324 ([C 1 -C 11 ] + , 20), 211 (12) , 169 (12), 105 (21), 75 (100) .
  • Less polar epimer (6b) a colorless oil, 30 mg yield (25%) .
  • Ma ⁇ ⁇ pectrum, tBDMSi derivative (m/z, % of related inten ⁇ ity) : 462 (0.04), 431 (0.35), 405 (20), 334 (4.7), 324 (2.8), 215 (4.2), 211 (3.0), 169 (6.0), 105 (26), 75 (100).
  • Thin layer chromatography was performed on aluminum sheets coated with silica gel 60 F 254, layer thickness 0.2 mm (Merck). HPLC was performed using a ⁇ Pora ⁇ il Si ⁇ 2 column, 3.9 x 300 mm for analy ⁇ i ⁇ and 7.8 x 300 mm for preparative ⁇ eparation (Water ⁇ ) , u ⁇ ing as eluent 0.7% i-PrOH in hexane for compounds (10a,b; numbers refer to Figure 2) and (13a,b), 0.3% i-PrOH in hexane for (lla,b) and (12a,b) .
  • Methyl 10(R,S)-hydroxy-ll(R) ,12(R)- epoxyeicosa-5Z,8Z,14Z-trienoates (10a,b) (unnatural epi eric HXB 3 ) were synthesized in an analogous manner to the natural epimers (Vasiljeva et al., Tetrahedron 49: 4099-4106, 1993).
  • the ratio between HXA3 and HXB3 benzoate ⁇ was establi ⁇ hed by the removal of benzoate group ⁇ (5 ml of 5% MeONa in MeOH, 2 hours, 20°C) followed by HPLC analysis (column ⁇ Porasil 3.9 x 300 mm, eluent 0.7% i-PrOH in hexane, detection at ⁇ 210 nm) .
  • Methyl (10S, 11R,12R) - threo-HxB 3 have 59% of the inverted methyl (10R, 11R, 12R) - erythro-HxB3 and 41% of the 6:1 mixture of methyl (8S,llR,12R)-syn- and (8S, 11R, 12R) -anti-HxA 3 whereas methyl (10R, 11R, 12R) -erythro-HxB 3 led to 90% of the methyl (10S, 11R, 12R) -t ⁇ reo-HxB 3 and 10% of the 1:6 mixture of methyl (8S, 11R, 12R) -syn- and (8R, 11R,12R) - nti-HxA3.
  • Ma ⁇ - ⁇ pectrum of the tBDMSi derivative (m/z, % of related inten ⁇ ity) : 448 ( [M - S]+, 0.22), 391 ([448 - t-Bu] + , 2.5), 307 ([C8-C20 - S] + , 100), 285 ([Cl-C8] + , 4.5), 175 ([307 - t-BDMSiOH] + , 6.1).
  • Human neutrophils were separated from fre ⁇ h human blood by dextran sedimentation followed by centrifugation on either a Ficoll-Hypaque gradient (Boyum, J. Clin. Invest. 2JL:77-98, 1968) or a discontinuous plasma-Percoll gradient (Downey et al., J. Appl. Physiol. S4 . :728-741, 1988) . Residual red blood cells were removed by lysis with NH4CI and centrifugation. Washed cells were maintained at room temperature in RPMI 1640 (Sigma Chemical Co., St.
  • HCO3 (buffered to pH 7.3 using 10 mM-Na Hepes) at IO 7 cell ⁇ /ml until assayed.
  • cell ⁇ [(1-2) X 10 7 cell ⁇ /ml] were ⁇ u ⁇ pended in medium containing NaCl (140 nM) , KC1 (5 nM) , MgCl 2 (1 nM) , CaCl 2 (1 nM) , Hepes (10 nM) and glucose (10 nM) , pH 7.3
  • the osmolarity of the media was adjusted to 290 + 5 mosm.
  • Intracellular free calcium was measured using the fluorescent indicator Indo-1-AM (Molecular Probes, Inc. , Eugene, OR) .
  • Indo-1-AM Molecular Probes, Inc. , Eugene, OR
  • the acetoxymethyl ester (AM) group on the dye allowed its penetration into the cell where estera ⁇ e ⁇ cleave it (by saponification) to release the free acid form which is trapped inside the cell.
  • Fmax-F All fluorescence values were measured relative to an MnCl 2 -quenched signal determined as follows: 250 nM is the K ( j of Indo-1-AM, F is the relative fluorescence measurement of sample. F max was determined by exposing cells to the calcium ionophore ionomycin (Sigma Chemical Co.) at 10 ⁇ 6 M final concentration. After F max was determined, MnCl 2 at 3 mM final concentration was added 5 to totally quench the Indo-1-AM signal and F ⁇ n was obtained. F m i n was obtained as follows: 1/12 x (F max - F Mn) + F Mn* Calibration was performed on each sample.
  • Hepoxilin analogs HXA 3 and other agonists were added to the cuvette as a 0 lOOOx concentrate in DMSO.
  • Hepoxilin analogs HA921-924 were tested fir ⁇ t at three concentrations (0.05, 0.1 and 0.5 ⁇ g/ l) in DMSO or the DMSO vehicle alone, followed five minutes later by each of the agonist ⁇ at one concentration, i.e. HxA 3 , 3 ⁇ g/ml; FMLP, 1 X 10" 9 M; LTB , 5 2 ng/ml; PAF, 1 ng/ l and thapsigargin, 10P ng/ml) .
  • IP showed that analogs HA-923 and HA-924 have IC 50 ⁇ of P.52 ⁇ M and P.3P ⁇ M, respectively, compared to IC 50 s for the
  • the hepoxilin ⁇ were tritated u ⁇ ing a modification of the procedure ⁇ described in Vasiljeva et al. Tetrahedron 4 ⁇ :4099, 1993 and Demin et al. Bioorg. Khim. ]6:1125, 1990.
  • the triacetylenic analog of HXB 3 (methyl 10(R/S)-hydroxy-11(S) ,12(S)-epoxy-5,8,14- eixo ⁇ atriynoate) wa ⁇ hydrogenated and tritiated using Lindlar catalyst in 4:1 (w/w) instead of 1:1 in the presence of quinoline and 100% tritium gas (1; numbers refer to the scheme shown in Figure 3) .
  • the resulting complex mixture contained two epimeric and over-tritiated products of [ 3 H 6 ]-HxB 3 that were separated using argentation thin layer chromotography.
  • the resulting [ 3 Hg]-HxB 3 epimers were separated from each other using straight phase HPLC.
  • the ratio between the epimers was 65:35 with a preference for the (R) epimer as in the starting triacetylene mixture (2)
  • the radiochemical purity of each epimer was >97% and the ⁇ pecific activity was approximately 169 Ci/mmol (2) .
  • Neutrophil suspensions (prepared as described previously) containing 2 x IO 6 cells in 1 ml total volume of as ⁇ ay medium (a ⁇ described in Example III) were added to [ 3 Hg]-HxA 3 (8S)-Me (32,000 cpm, specific activity 169 Ci/mmol) in the absence or presence of 0.5 or 1 ⁇ g of unlabelled hepoxilin to measure non specific binding.
  • 0.5 ⁇ g of cyclopropyl analogs HA-923 or HA-924 was added to the tritiated hepoxilin. The tubes were incubated at 37°C for 1 hour.
  • the reaction mixture was rapidly filtered through Whatman GF/B glass fiber filters (Whatman Bio ⁇ ystems, Maidstone, England) that had been prewashed with the a ⁇ ay medium.
  • the tube ⁇ were rin ⁇ ed three time ⁇ with ice-cold a ⁇ ay medium.
  • the radioactivity retained on the filter ⁇ was counted in 10 ml of ECOLITE scintillation fluid (ICN, St. Laurent, Quebec) in a Beckman (Model LS 3800) scintillation spectrophotometer (Beckman, Irvine, CA) .
  • Figure 4 illustrates that specific binding of hepoxilin A 3 occurs in human neutrophils and that hepoxilin analogs HA-923 and HA-924 inhibit the binding of binding of tritiated hepoxilin A 3 .

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  • Steroid Compounds (AREA)

Abstract

Composés présentant la structure générale (a) ou (b), dans laquelle X représente O, Cn, NH ou S, où n vaut 1, 2, 3 ou 4; R1 représente OH, CH3, CH2OH, N3 ou CH2N3; R3 représente H ou CH3; R5 représente Y-R2, où Y représente une chaîne à six carbones renfermant éventuellemenet jusqu'à trois liaisons doubles ou triples ou un mélange de liaisons doubles et triples jusqu'à un maximum de trois; R2 représente alkyle C1-10 OH, alkyle C1-10 N3 ou COOR4, où R4 représente H, alkyle C1-10 éventuellement ramifié (les radicaux d'alkyle substitué compris), cycloalkyle, de préférence cycloalkyle C5 ou C6, ou un radical aryle pentagonal ou hexagonal (les radidaux d'aryle substitué compris), c'est-à-dire que R2 représente COOH ou un ester de R4; R6 représente une chaîne à sept carbones renfermant éventuellement jusqu'à trois liaisons doubles ou triples ou un mélange de liaisons doubles et triples jusqu'à un maximum de trois; et (c) représente une liaison dimple, double ou triple. Ces composés sont des analogues des hépoxilines et servent à moduler l'activité de celles-ci, par exemple dans le but de limiter l'inflammation ou d'autres processus dus aux taux de calcium intracellulaire.
PCT/IB1994/000085 1993-03-29 1994-03-28 Analogues d'hepoxiline utilisables comme agents anti-inflammatoires WO1994022848A1 (fr)

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AU65117/94A AU6511794A (en) 1993-03-29 1994-03-28 Hepoxilin analogs useful as anti-inflammatory agents
DE69411716T DE69411716T2 (de) 1993-03-29 1994-03-28 Hepoxilin analoga verwendbar als entzündungshemmende mittel
EP94912660A EP0691963B1 (fr) 1993-03-29 1994-03-28 Analogues d'hepoxiline utilisables comme agents anti-inflammatoires

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US3832493A 1993-03-29 1993-03-29
US08/038,324 1993-03-29

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WO2001010422A3 (fr) * 1999-08-04 2001-08-23 Cecil R Pace-Asciak Analogues d'hepoxiline
WO2002038157A3 (fr) * 2000-11-09 2003-02-27 Hospital For Sick Children Inhibiteurs de la formation et de l'activite de la thromboxane
US6673785B1 (en) 1999-08-04 2004-01-06 Cecil R. Pace-Asciak Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinflammatory agents
WO2004104022A3 (fr) * 2003-05-16 2005-05-12 Gen Hospital Corp Compositions contenant un facteur chimio-tactique epithelial pathogene (eicosanoide et hepoxiline a3), leurs inhibiteurs et leurs procedes d'utilisation
WO2006106438A3 (fr) * 2005-01-11 2007-05-18 Evolva Sa Procedes de modulation de l'activite de ppar
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JP2023536686A (ja) * 2020-07-30 2023-08-29 フイルメニツヒ ソシエテ アノニム ジエンの調製方法

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WO1996011908A1 (fr) * 1994-10-13 1996-04-25 Peptide Technology Limited Acides gras polyinsatures modifies
US6376688B1 (en) 1994-10-13 2002-04-23 Peptide Technology Limited Modified polyunsaturated fatty acids
WO2001010422A3 (fr) * 1999-08-04 2001-08-23 Cecil R Pace-Asciak Analogues d'hepoxiline
US6673785B1 (en) 1999-08-04 2004-01-06 Cecil R. Pace-Asciak Use of hepoxilins or hepoxilin analogs as antidiabetics, antiinflammatory agents
WO2002038157A3 (fr) * 2000-11-09 2003-02-27 Hospital For Sick Children Inhibiteurs de la formation et de l'activite de la thromboxane
US8071654B2 (en) 2000-11-09 2011-12-06 Pace-Asciak Cecil R Inhibitors of thromboxane formation and action
WO2004104022A3 (fr) * 2003-05-16 2005-05-12 Gen Hospital Corp Compositions contenant un facteur chimio-tactique epithelial pathogene (eicosanoide et hepoxiline a3), leurs inhibiteurs et leurs procedes d'utilisation
WO2006106438A3 (fr) * 2005-01-11 2007-05-18 Evolva Sa Procedes de modulation de l'activite de ppar
US8710252B2 (en) 2006-04-19 2014-04-29 Cecil Pace-Asciak Hepodxilin analog enantiomers
WO2019010414A1 (fr) * 2017-07-07 2019-01-10 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Dérivés d'acides gras et utilisation associée
CN110869354A (zh) * 2017-07-07 2020-03-06 美国政府(由卫生和人类服务部的部长所代表) 脂肪酸衍生物及其用途
KR20200027545A (ko) * 2017-07-07 2020-03-12 더 유나이티드 스테이츠 오브 어메리카, 애즈 리프리젠티드 바이 더 세크러테리, 디파트먼트 오브 헬쓰 앤드 휴먼 서비씨즈 지방산 유도체 및 이의 용도
JP2020526505A (ja) * 2017-07-07 2020-08-31 ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ 脂肪酸誘導体およびこれらの使用
US11555021B2 (en) 2017-07-07 2023-01-17 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Fatty acid derivatives and their use
KR102725883B1 (ko) * 2017-07-07 2024-11-01 더 유나이티드 스테이츠 오브 어메리카, 애즈 리프리젠티드 바이 더 세크러테리, 디파트먼트 오브 헬쓰 앤드 휴먼 서비씨즈 지방산 유도체 및 이의 용도
JP2023536686A (ja) * 2020-07-30 2023-08-29 フイルメニツヒ ソシエテ アノニム ジエンの調製方法

Also Published As

Publication number Publication date
DE69411716D1 (de) 1998-08-20
US5616607A (en) 1997-04-01
CA2159584A1 (fr) 1994-10-13
EP0691963B1 (fr) 1998-07-15
EP0691963A1 (fr) 1996-01-17
AU6511794A (en) 1994-10-24
DE69411716T2 (de) 1999-03-11
ATE168372T1 (de) 1998-08-15

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